40 publications mentioning rno-mir-26b

Open access articles that are associated with the species Rattus norvegicus and mention the gene name mir-26b. Click the [+] symbols to view sentences that include the gene name, or the word cloud on the right for a summary.

1

To further investigate the therapeutic role of miR-26b in human tissues, we transfected hPASMCs with miR-26b mimics, CTGF or CCND1 specific siRNA, and examined the inhibitory effect of miR-26b on the expression levels of CTGF and CCND1 in hPASMCs, finding that the effect of specific siRNA was similar to that of miR-26b with respect to the inhibition of the expression of the designed target gene, whereas only miR-26b could suppressed the expression of both CTGF and CCND1 (Figure 4).

Taken together, we showed that PASMCs from monocrotaline -induced pulmonary artery remo deling are different from the normal controls in their microRNA repertoire, and miR-26b, a microRNA significantly downregulated in pulmonary artery remo deling, contributes to the development of PAH via releasing the inhibition of its two target genes, CTGF and CCND1, both of which have been repeatedly reported to be involved in the pathogenesis of the disease.

The discovery of the downregulation of miR-26b in pulmonary artery remo deling, as well as its regulation of its target genes, CTGF and CCND1, may have important implications in our understanding of the molecular mechanisms underlying PAH, and may also lead to the development of new therapeutic interventions in this devastating and life-threatening disease.

Considering the reports that the upregulation of CTGF and CCND1 substantially contributes to the development of pulmonary vascular remo deling, as well as the observation that miR-26b significantly suppresses CTGF and CCND1 expression, we next evaluated its effect on monocrotaline -induced pulmonary vascular remo deling, and determined the influence of miR-26b/EXGEN500 complex on the expression of CTGF and CCND1 in parallel with CTGF and CCND1 shRNAs in monocrotaline -treated rats.

Figure 4 A. Effect of introduction of anti-CTGF siRNA, anti-CCND1 siRNA, has-miR-26b mimics and control on the mRNA expression level of CTGF in hPASMCs (p<0.01); B. lower panel: Effect of introduction of anti-CTGF siRNA, anti-CCND1 siRNA, has-miR-26b mimics and control on the protein expression level of CTGF in hPASMCs, as determined by western blot; upper panel: densitometric analysis of the western blot results underneath (p<0.01); C. Effect of introduction of anti-CTGF siRNA, anti-CCND1 siRNA, has-miR-26b mimics and control on the mRNA expression level of CCND1 in hPASMCs (p<0.01); D. lower panel: Effect of introduction of anti-CTGF siRNA, anti-CCND1 siRNA, has-miR-26b mimics and control on the protein expression level of CCND1 in hPASMCs, as determined by western blot; upper panel: densitometric analysis of the western blot results underneath (p<0.01).

Furthermore, the most significantly down-regulated miRNAs, miR-26b, was selected for further functional analysis due to its well documented role in regulating human cell proliferation [30, 31], as well as the predicted function to suppress the expression of CTGF and CCND1(www.

A. Effect of introduction of anti-CTGF siRNA, anti-CCND1 siRNA, has-miR-26b mimics and control on the mRNA expression level of CTGF in hPASMCs (p<0.01); B. lower panel: Effect of introduction of anti-CTGF siRNA, anti-CCND1 siRNA, has-miR-26b mimics and control on the protein expression level of CTGF in hPASMCs, as determined by western blot; upper panel: densitometric analysis of the western blot results underneath (p<0.01); C. Effect of introduction of anti-CTGF siRNA, anti-CCND1 siRNA, has-miR-26b mimics and control on the mRNA expression level of CCND1 in hPASMCs (p<0.01); D. lower panel: Effect of introduction of anti-CTGF siRNA, anti-CCND1 siRNA, has-miR-26b mimics and control on the protein expression level of CCND1 in hPASMCs, as determined by western blot; upper panel: densitometric analysis of the western blot results underneath (p<0.01).

In addition, miR-26b almost completely restored the monocrotaline -induced up-regulation of CTGF and CCND1, while shRNA only partially lowered the expression of its designed target, as shown in Supplementary Figure S2 and S3.

To elucidate how miRNAs are involved in PAH pathogenesis, we investigated and compared global miRNA expression profiles in PASMCs isolated from monocrotaline -treated rats and normal control using microarray and selected the most significantly down-regulated miRNAs, miR-26b, for further functional analysis due to its documented role in regulating human cell proliferation [27], as well as the virtual function to suppress the expression of CTGF and CCND1 predicted by online microRNA databases such as www.

In this study, we found that overexpression of miR-26b, but not the control, substantially repressed the activity of luciferase fused with 3′-UTR of CTGF and CCND1 respectively, but had minimal effect on the luciferase activity fused with mutated 3′-UTRs, as shown in Figure 1. In addition, we found that relative expression of miR-26b in the rPASMCs isolated from monocrotaline -treated rats was significantly downregulated compared with the control.

Figure 2 A. Determination and comparison of expression of rno-miR-26b between rPASMCs harvested from monocrotaline -treated and normal saline -treated rats (p<0.01); B. Determination and comparison of mRNA expression level of CTGF in rPASMCs harvested from the rats treated with normal saline, monocrotaline+NCshRNA, CTGF shRNA, CCND1 shRNA, and miR-26b (p<0.01); C. Determination and comparison of mRNA expression level of CCND1 in rPASMCs harvested from the rats treated with normal saline, monocrotaline+NCshRNA, CTGF shRNA, CCND1 shRNA, and miR-26b (p<0.01); D. Upper panel: Densitometry analysis of the western blotting results to show the relative protein levels of CCND1 in rPASMCs harvested from the rats treated with normal saline, monocrotaline+NCshRNA, CTGF shRNA, CCND1 shRNA, miR-26b; Lower panel: Results of western blot (p<0.01); E. Upper panel: Densitometry analysis of the western blotting results to show the relative protein levels of CTGF in rPASMCs harvested from the rats treated with normal saline, monocrotaline+NCshRNA, CTGF shRNA, CCND1 shRNA, miR-26b; Lower panel: Results of western blot (p<0.01).

MiR-26b has been shown to be able to suppress the proliferation of human multiple myeloma cells via targeting a number of candidate genes including CTGF [39]; Meanwhile, Yang et al demonstrated in their study about human breast cancer that miR-26b inhibited breast cancer progression through modulating Fra-1 proto-oncogene [27].

In the present study, we found that downregulation of miR-26b was responsible for the upregulation of CTGF and CCND1 in monocrotaline -induced pulmonary artery remo deling, and intratracheal administration of miR-26b could almost completely restore the pulmonary artery remo deling in monocrotaline -treated rats.

Site-directed mutagenesis of the miRNAs binding sites in the 3′UTRs was performed using Site-Directed Mutagenesis Kit (SBS Genetech, Beijing, China) and named as mutant 3′UTRs, as shown in Figure 1. rPASMCs grown in a 48-well plate were co -transfected with 400 ng of either individual miR-26b, 40 ng of the firefly luciferase reporter plasmid including the 3′-UTR of the target gene, and 4 ng of pRL-TK, a plasmid expressing rellina luciferase (Promega, Madison, WI, USA).

As shown in Figures 2B-2E, the treatment with miR-26b/EXGEN500 complex significantly attenuated both CTGF and CCND1 mRNA/protein expression in rat pulmonary vessels compared with the control, while the two shRNAs specifically downregulated its designed target with minimal effect on the other one.

A. Determination and comparison of expression of rno-miR-26b between rPASMCs harvested from monocrotaline -treated and normal saline -treated rats (p<0.01); B. Determination and comparison of mRNA expression level of CTGF in rPASMCs harvested from the rats treated with normal saline, monocrotaline+NCshRNA, CTGF shRNA, CCND1 shRNA, and miR-26b (p<0.01); C. Determination and comparison of mRNA expression level of CCND1 in rPASMCs harvested from the rats treated with normal saline, monocrotaline+NCshRNA, CTGF shRNA, CCND1 shRNA, and miR-26b (p<0.01); D. Upper panel: Densitometry analysis of the western blotting results to show the relative protein levels of CCND1 in rPASMCs harvested from the rats treated with normal saline, monocrotaline+NCshRNA, CTGF shRNA, CCND1 shRNA, miR-26b; Lower panel: Results of western blot (p<0.01); E. Upper panel: Densitometry analysis of the western blotting results to show the relative protein levels of CTGF in rPASMCs harvested from the rats treated with normal saline, monocrotaline+NCshRNA, CTGF shRNA, CCND1 shRNA, miR-26b; Lower panel: Results of western blot (p<0.01).

By searching online microRNA databases, we identified both CTGF and CCND2 as potential targets of miR-26b, the most down-regulated one.

Treatment with monocrotaline significantly downregulated miR-26b expression in rats.

Considering the previous reports that miR-26b inhibited human cell proliferation, and the observation that miR-26b substantially suppressed the expression of CTGF and CCND1, we subsequently evaluated the efficacy of miR-26b in the treatment of monocrotaline -induced pulmonary artery remo deling in parallel with CTGF or CCND1 shRNA, and found miR-26b, CTGF shRNA, or CCND1 shRNA significantly decreased the pulmonary vessel wall thickness (42%, 45%, 20%, of monocrotaline treated, respectively) in monocrotaline -treated rats.

Figure 2A showed that relative expression of miR-26b in the rPASMCs isolated from monocrotaline -treated rats was significantly down-regulated to about 20% compared with the control (*P<0.01).

In vitro analysis of the inhibitory effect of miR-26b on the expression of CTGF and CCND1 in comparison with CTGF or CCND1 specific siRNA in hPASMCs.

We reasoned that miR-26b may exert protective effect via inhibiting both CTGF and CCND1, or possibly involved some other PAH-related genes which are not necessarily a complete match, and therefore not predictable by the online target predicting tool.

Furthermore, we showed in this study that the miR-26b also significantly blocked monocrotaline -induced upregulation of α-SM-actin in rats, and the shRNAs could only partially restored it (Supplementary Figure S4).

In this study, a similar delivery efficiency and a better efficacy of miR-26b/EXGEN500 were exhibited in the treatment of same disease, and this may shed a light on the development of therapeutic tool in treat PAH in human.

Effect of CTGF siRNA, CCND1 siRNA and miR-26b on the expression of CTGF and CCND1 in rPASMCs.

The results demonstrated that even though CTGF or CCND1 shRNA treatment successfully inhibited the pulmonary vascular remo deling induced by monocrotaline, but neither of them could completely restore the pulmonary artery remo deling induced by monocrotaline, while miR-26b could almost completely restore it.

We found that overexpression of miR-26b, but not the control mimics, substantially repressed the activity of luciferase fused with wild-type 3′-UTR of CTGF and CCND1, respectively, but had minimal effect on the luciferase activity fused with mutated 3′-UTR of CTGF or CCND1, as shown in Figures 1D, 1E (*P<0.01).

Louis, MO, USA) and negative control shRNA (as therapeutic control); Group III: Treated with monocrotaline and CTGF shRNA; Group IV: Treated with monocrotaline and CCND1 shRNA; Group V: Treated with monocrotaline and miR-26b (No difference was identified regarding the observed parameters such as cell proliferation, vascular thickness, the expression of CTGF, CCND1,α-SM actin, or cell cycle progression between the rats treated with or without normal saline, and between the rats treated with monocrotaline and monocrotaline+ NCshRNA (Negative control shRNA), as presented in our previous studies, so we narrowed down the original seven groups to five experimental groups in this study).

Figure 1 A. The “seed sequence” in the 3′ UTR of the target gene of rno-miR-26b is highly conserved among the species including, but not limited to, Rno, Has, Ptr, Mml, Oga, Tbe, and Mmu; B. Schematic comparison between rno-miR-26b and the wild-type (upper sequence)/mutated (lower sequence) 3′UTR of CTGF with “seed sequence” highlighted; C. Schematic comparison between rno-miR-26b and the wild-type (upper sequence)/mutated (lower sequence) 3′UTR of CCND1 with “seed sequence” highlighted; D. The relative luciferase activity in the rPASMCs transfected with both wild-type 3′UTR of CTGF and rno-miR-26b is significantly lower than the controls (p<0.01); E. The relative luciferase activity in the rPASMCs transfected with both wild-type 3′UTR of CCND1 and rno-miR-26b is significantly lower than the controls (p<0.01).

Furthermore, we validated CTGF and CCND1 as effective targets of miR-26b by using a luciferase reporter system.

Effect of MCT, CTGF shRNA, CCND1 shRNA and miR-26b on the expression of CTGF and CCND1 in rPASMCs.

These results indicated that treatment with miR-26b substantially induced G1 cell cycle arrest in rPASMCs, and its inhibitory effect was stronger than that of in the groups treated with CTGF or CCND1 shRNA.

Flow cytometry analysis of the inhibitory effect of miR-26b on cell cycle progression in comparison with CTGF or CCND1 shRNAs in rPASMCs.

Sequence comparison between miR-26b and 3′UTR of CTGF and CCND1.

A. Flow cytometric determination of cell cycle status in rPASMCs harvested from the rats treated with normal saline; B. Flow cytometric determination of cell cycle status in rPASMCs harvested from the rats treated with monocrotaline+NCshRNA; C. Flow cytometric determination of cell cycle status in rPASMCs harvested from the rats treated with monocrotaline and CTGF shRNA; D. Flow cytometric determination of cell cycle status in rPASMCs harvested from the rats treated with monocrotaline and CCND1 shRNA; E. Flow cytometric determination of cell cycle status in rPASMCs harvested from the rats treated with monocrotaline and rno-miR-26b.

The results demonstrated that even though CTGF or CCND1 shRNA treatment successfully attenuated the pulmonary vascular remo deling induced by monocrotaline, nevertheless neither of them could completely restore the pulmonary artery remo deling, which can be done by introduction of miR-26b alone.

For gene delivery in vivo, plasmid -based CTGF or CCND1 shRNA, miR-26b, or its negative control were mixed with EXGEN500 and 5% glucose (Fermentas Biotechnology Co.

Effect of MCT, CTGF shRNA, CCND1 shRNA and miR-26b on the cell cycle status in rPASMCs.

Meanwhile, the treatment with CTGF or CCND1 shRNA, and miR-26b significantly decreased the pulmonary vessel wall thickness (42%, 45%, 20% of monocrotaline -treated, respectively) in monocrotaline -treated rats (H&E staining, images not shown).

Briefly, the oligonucleotides for CTGF or CCND1 shRNAs, miR-26b or the negative control were synthesized chemically or PCR-amplified, and linked into the pGPU6/GFP vector which contains a mouse U6 RNA polymerase III promoter.

Figure 3 A. Flow cytometric determination of cell cycle status in rPASMCs harvested from the rats treated with normal saline; B. Flow cytometric determination of cell cycle status in rPASMCs harvested from the rats treated with monocrotaline+NCshRNA; C. Flow cytometric determination of cell cycle status in rPASMCs harvested from the rats treated with monocrotaline and CTGF shRNA; D. Flow cytometric determination of cell cycle status in rPASMCs harvested from the rats treated with monocrotaline and CCND1 shRNA; E. Flow cytometric determination of cell cycle status in rPASMCs harvested from the rats treated with monocrotaline and rno-miR-26b.

Plasmids containing miR-26b, CTGF or CCND1 shRNA, or the control were mixed with EXGEN500/5% Glucose prior to intratracheal administration.

2

In order to clarify the relationship between miR-26b and IL-6, we used miR-26b mimics that increased miR-26b expression (Figure 2D) and an miR-26b inhibitor that inhibited miR-26b function (there was no statistical difference in miR-26b expression; Figure 2D) to transfect the microglial cells and to examine their effect on IL-6 expression.

Yet, because miRNA can have many targets and the downstream target gene of miR-26b could also be MAPK and so on, miR-26b overexpression may also change the expression of these targets, thus affecting cellular function.

Increased miR-26b expression by its mimics in cultured microglia reduced the IL-6 level, while the miR-26b inhibitor increased IL-6 expression, suggesting that miR-26b activation is negatively correlated to IL-6 expression.

Figure 2OGD induced changes of IL-6 expression in microglia, which was associated with downregulation of miR-26b.

MiRNA-26b has mostly been studied in cancer, and it has been found that miR-26b is downregulated in breast cancer and that it can inhibit cellular proliferation (Li J. et al., 2014).

In addition, activated microglia could downregulate miR-26b expression.

Furthermore, both and BCCAO reduced miR-26 expression but increased IL-6 expression.

Figure 8Increased miR-26b expression inhibited neuronal damage and improved cognitive impairment of the VCI mo del.

In primary microglia, could also induce the release of large amounts of inflammatory cytokines, including IL-6. Previously, it has been shown that IL-6 and other inflammatory factors are increased after treatment in BV-2 cells; in addition, the expression of many miRNAs is altered, including decreased miR-26b expression (Carlson et al., 1999; Zhang et al., 2012, 2015; Prinz and Priller, 2014).

Moreover, increased miR-26b expression in the hippocampal CA1 area in the VCI rat mo del could inhibit the activation of microglia and the production of IL-6 as well as reduce neuronal apoptosis, thus alleviating the cognitive impairment.

Moreover, previous studies have shown that miRNA-26b is downregulated when exposed to oxygen-glucose deprivation (OGD; Zhang et al., 2012).

However, increased miR-26b expression could attenuate microglial activation, inflammatory reactions, and cognitive function, which may occur via IL-6 regulation.

MiRNA-26b inhibits cellular proliferation by targeting CDK8 in breast cancer.

Figure 3IL-6 is the target gene of miR-26b regulation.

These results indicate that miR-26b may inhibit transcription of IL-6 by binding to the IL-6 mRNA 3′-UTR, resulting in reduced IL-6 production.

However, increased miR-26b expression in the hippocampal CA1 area could reduce brain inflammation as well as neuronal damage.

Our previous study found that the IL-6 3′-UTR region might have an miR-26b binding site, suggesting that IL-6 may be a target gene of miR-26b (Zhang et al., 2015).

Induced Microglial Activation and Reduced miR-26b Expression In vitro, we used to induce hypoxia in microglia.

We showed that the microglial cell supernatant induced neuronal apoptosis; however, the miR-26b mimics alleviated the neuronal apoptosis, while the miR-26b inhibitor aggravated the neuronal apoptosis in both primary neurons (Figures 4A,B) and SH-SY5Y cells (Figures 4C,D).

In contrast, the miR-26b inhibitor significantly increased the IL-6 level (Figure 2E).

The miR-26b mimics (5′-UUCAAGUAAUUCAGGAUAGGU-3′ and 5′-CUAUCCUGAAUUACUUGAAUU-3′) and the negative controls (5′-UUCUUCGAACGUGUCACGUTT-3′ and 5′- ACGUGACACGUUCGGAGAATT-3′) as well as the miR-26b inhibitor (5′-ACCUAUCCUGAAUUACUUGAA-3′) and the negative control (5′-CAGUACUUUGUGUAGUACAA-3′) were synthesized by Genepharma (Shanghai, China).

IL-6 May be the Target of miR-26b.

The reduction of miR-26b expression was related to the activation of microglia, the generation of IL-6, and the neurotoxic effects of activated microglia.

Meanwhile, we found that significantly reduced the miR-26b expression after reoxygenation for 3 h and 6 h after treatment (Figure 2C).

The activated microglia induced by could increase neuronal apoptosis, which was reversed by the increase of miR-26b expression.

The miR-26 expression was significantly increased at 3 or 7 days after the LV-26b injection (Figure 7B).

To increase the level of miR-26b expression, we stereotactically injected miR-26b lentiviral vector (LV-26b) into the hippocampal CA1 area at day 2 after the VCI rat mo del was established.

However, it is unclear how miRNA-26b regulates the microglial inflammatory response in hypoxia/ischemia and how it affects the development of VCI.

OGD Induced Microglial Activation and Reduced miR-26b Expression.

In the VCI animal mo del, we found that the microglia were activated, IL-6 production was increased, and the expression of miR-26b was reduced in the hippocampal CA1 area; these data were consistent with the experimental results.

After, we found that IL-6 expression was significantly reduced in the miR-26b mimic groups, compared to that in the control group.

Meanwhile, the miR-26b expression was significantly reduced in the BCCAO group, compared to the sham group (Figure 6G).

However, the regulation of miRNA-26b in microglial activation in VCI remains unclear.

MiR-26b Could Inhibit Neuronal Apoptosis Induced by Microglial Activation in OGD.

MiR-26b Could Inhibit Neuronal Apoptosis Induced by Microglial Activation inMicroglia released large amounts of inflammatory cytokines after.

MiR-26b Overexpression Could Alleviate Damage in the CA1 Area and Improve Learning and Memory Abilities in the VCI Mo del.

In both 293T cells (Figure 3B) and BV-2 cells (Figure 3C), we found that the luciferase activity was inhibited in the wild-type by the miR-26b mimics, compared to that in the control group, while there was no significant difference in the luciferase activity of the mutant type between the miR-26b mimic groups and the control group.

The Effect of miR-26b on Microglia-Activated Toxicity, Which Was Partially Dependent on IL-6. Microglial Activation, Neuronal Damage, and miR-26b Changes in the VCI Mo del.

Figure 5The effect of miR-26b on microglial neurotoxicity depended on the involvement of IL-6. (A–B) Microglia were transfected with IL-6 siRNA or negative controls; after and reoxygenation for 48 h, IL-6 mRNA was detected by PCR (A) and the concentration of IL-6 was detected by ELISA (B).

To further clarify the role of IL-6 in regulating miR-26b in microglial neurotoxicity, we transfected IL-6 siRNA (siIL-6) into microglia and found that the IL-6 mRNA level (Figure 5A) and IL-6 content (Figure 5B) were significantly reduced after, compared to the control, which imitated the effect of miR-26b, and the MCM, after transfection with siIL-6, could also lower the mortality of neurons after (Figure 5C).

The cells were cotransfected with the miR-26b mimics and the IL-6 mRNA 3′-UTR luciferase plasmid, or the miR-26b negative control and the IL-6 mRNA 3′-UTR luciferase plasmid by using the transfection reagent Lipofectamine 2000.

Furthermore, we used a dual luciferase reporter system to demonstrate that miR-26b could bind to the IL-6 mRNA 3′-UTR rather than the mutant.

Briefly, before transfection, the cell culture medium was changed to new complete growth medium, and the cells were incubated for 1 h. The miR-26b or siIL6 oligonucleotides and reagent were added to Pepmute transfection buffer, and the mixture was incubated at room temperature for 15 min before it was added to the cells.

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miRNA Host gene Function of host gene miR-923 UNC45B UNC45B plays a role in myoblast fusion and sarcomere organization miR-126 EGFL7 blood vessel development; angiogenesis; and vasculogenesis miR-26b CTDSP1 n/a miR-199a DNM2/DNM3 filopodium formation; centronuclear myopathy; growth and development of megakaryocytes miR-214 DNM3 filopodium formation; centronuclear myopathy; growth and development of megakaryocytes miR-499 MYH7B cardiac muscle, striated muscle contraction, striated muscle thick filament miRNA regulates gene expression by binding and modulating the translation of specific miRNAs.

On day 7, miR-31, miR-214, miR-199a-5p, and miR-199a-3p were up-regulated, whereas miR-181c, miR-29b, miR-26b, miR-181d, mir-126, mir-499-5p, and miR-1 were down-regulated.

miRNA Host gene Function of host gene miR-923 UNC45B UNC45B plays a role in myoblast fusion and sarcomere organization miR-126 EGFL7 blood vessel development; angiogenesis; and vasculogenesis miR-26b CTDSP1 n/a miR-199a DNM2/DNM3 filopodium formation; centronuclear myopathy; growth and development of megakaryocytes miR-214 DNM3 filopodium formation; centronuclear myopathy; growth and development of megakaryocytes miR-499 MYH7B cardiac muscle, striated muscle contraction, striated muscle thick filament Microarray data mining and differential analyses resulted in 17 significantly deregulated miRNAs associated with AMI (Table 1, Figure 1).

Some of the deregulated miRNAs (miR-181, miR-26, miR-1, mir-29, miR-214, miR-126, and miR-499) are reported to be related to hypoxia, cell development, and cell growth [1, 5, 7, 25].

4

Among the miRNAs examined, 79 miRNAs (24%) responded to the hyperandrogenic condition and interestingly, 80% of which were upregulated compared to the control group supporting the notion that hyperandrogenic condition down-regulates androgen receptors in the granulosa cells [35] which could be mediated by these upregulated miRNAs (rno-miR-379*, rno-let-7d, rno-miR-24, rno-miR-673, rno-miR-26b, rno-miR-335, rno-miR-382*, rno-miR-412, rno-miR-99a*, rno-miR-543, rno-miR-674-3p, rno-miR-409-3p).

In addition to miR-221/222, several studies also highlighted the differential regulation of let-7d, let-7f, miR-25 and miR-26b in prostate and breast cancer, as well as in leukemia by the estrogen receptor pathways and that their expression was up-regulated in ERα -positive cells [50- 52].

A list of differentially expressed miRNAs (Fold change ≥ 2 and their corresponding P value) is presented in Figure  4. Beside this group, miRNAs which were also highly abundant in DHT -treated ovaries are rno-miR-221, rno-miR-222, rno-miR-25, rno-miR-26b, rno-miR-379*, rno-let-7d, rno-miR-24, rno-miR-673, rno-miR-26b, rno-miR-335, rno-miR-382*, rno-miR-412, rno-miR-99a*, rno-miR-543, rno-miR-674-3p, rno-miR-409-3p.

Among the fourteen miRNAs mapped to the ingenuity databases, twelve (rno-let-7d, rno-miR-132, rno-miR-182, rno-miR-183, rno-miR-184, rno-miR-21, rno-miR-221, rno-miR-24, rno-miR-25, rno-miR-26b, rno-miR-31 and rno-miR-96) had 171 experimentally validated targets.

5

Thus, miRNA families (e. g., miR-1 and miR-122) that are specifically or highly expressed in any one of the 3 tissues, or miRNAs that are expressed ubiquitously (e. g., let-7 and miR-26) in all 3 tissues, show a far greater frequency than other miRNAs.

The observation that miR-22, miR-26b, miR-126, miR-29c and miR-30c are ubiquitously expressed in 14 different tissues of pig is interesting.

Additionally, many other miRNAs, such as let-7, miR-98, miR-16, miR22, miR-26b, miR-29c, miR-30c and miR126, were also expressed abundantly in thymus (Figure 3).

miR-22, miR-26b, miR-29c, miR-30c and miR-126 exhibited almost similar expression patterns in all tissues examined (Figure 3B).

For instance, let-7 is represented by 445 reads and miR-26 by 177 reads (Tables 1 and 2), and these two miRNAs are ubiquitously expressed in the heart, liver and thymus (Figure 3A and 3B).

miR-22, miR-26b, miR-29c and miR-30c showed ubiquitous expression in diverse tissues.

6

Finally, we screened two miRNAs: miR-26 highly expressed in the CNS (Smirnova et al., 2005), and miR-146a, an important regulator of innate immune responses in microglia (Rom et al., 2010; Saba et al., 2012; Ponomarev et al., 2013).

Although they did not reach statistical significance, we found that compared to expression levels in microglia, miR-26 tended to be 2–3-fold more highly expressed in neurons (P = 0.051) and astrocytes (P = 0.080).

miR-26 expression levels were comparable in astrocytes and neurons (P = 0.696).

Because miR-26 is reported to be preferentially expressed in astrocytes (Smirnova et al., 2005), we were surprised to find it equally detectable in astrocytes and neurons.

However, the astrocyte-specific expression of miR-26 was determined in differentiated murine stem cells in vitro (Smirnova et al., 2005).

1 TGC TCG CGA CCT CAA TGT A GGT AGA AGC AGA GCG GAC TT JMJD8 Jmjd8 NM_001014116.1 TGG ACG ATT CGG TCT GCT TT ACT CTG TTT CCA TCC CCC TTC Mina53 Mina53 NM_153309.2 ATG CCA AAG AAA GTG AAG CCC GTA GCT CCT CTT TCA CCT GCT PHF2 Phf2 NM_001107342.1 TCA GAC ACC AGA ATG TCC AGC TCG GGC CAG TAG TTT TCC AC PHF8 Phf8 NM_001108253.1 TTT GGG ACC GTG GAC GTT T GTC AGA AAG GCA GCA ACA AGC UTX Kdm6a NM_009483.1 CCA CCC TGC CTA GCA ATT CA CCA CCT GAG GTA GCA GTG TG UTX Uty NM_009484 ATT ATC TCT CAC TAC TGC TGC CC CGA AGA AGC TGC TGT CTA ATC CAC snoRNA135/Snord65 NR_028541.1 AGT ACT TTT TGA ACC CTT TTC CA snoRNA234/Snord70 NR_028554.1 TTA ACA AAA ATT CGT CAC TAC CA mir-26 NR_029742.1 GGT TCA AGT AAT CCA GGA TAG GCT mir-146a NR_031892.1 TGA GAA CTG AAT TCC ATG GGT T were performed on delta C [T] values using Sigma Plot 11.0 software.

1 TGC TCG CGA CCT CAA TGT A GGT AGA AGC AGA GCG GAC TT JMJD8 Jmjd8 NM_001014116.1 TGG ACG ATT CGG TCT GCT TT ACT CTG TTT CCA TCC CCC TTC Mina53 Mina53 NM_153309.2 ATG CCA AAG AAA GTG AAG CCC GTA GCT CCT CTT TCA CCT GCT PHF2 Phf2 NM_001107342.1 TCA GAC ACC AGA ATG TCC AGC TCG GGC CAG TAG TTT TCC AC PHF8 Phf8 NM_001108253.1 TTT GGG ACC GTG GAC GTT T GTC AGA AAG GCA GCA ACA AGC UTX Kdm6a NM_009483.1 CCA CCC TGC CTA GCA ATT CA CCA CCT GAG GTA GCA GTG TG UTX Uty NM_009484 ATT ATC TCT CAC TAC TGC TGC CC CGA AGA AGC TGC TGT CTA ATC CAC snoRNA135/Snord65 NR_028541.1 AGT ACT TTT TGA ACC CTT TTC CA snoRNA234/Snord70 NR_028554.1 TTA ACA AAA ATT CGT CAC TAC CA mir-26 NR_029742.1 GGT TCA AGT AAT CCA GGA TAG GCT mir-146a NR_031892.1 TGA GAA CTG AAT TCC ATG GGT T Statistical analyses were performed on delta C [T] values using Sigma Plot 11.0 software.

7

MiR-504 mediates expression of the dopamine D1 receptor [26] and miR-26b is reported to negatively regulate brain-derived neurotrophic factor (BDNF) expression at post-transcriptional level [27].

Repeated cocaine exposure produced an increase in expression of miR-26b and a decrease in expression of miR-191 in CCA rats (p < 0.05 vs.

Some miRNAs which have been previously reported to be involved in brain disorders and drug abuse, including miR-133b, miR-134, miR-181c, miR-191, miR-22, miR-26b, miR-382, miR-409-3p and miR-504, were found to be changed in their expression following repeated cocaine exposure and subsequent abstinence from cocaine treatment.

To confirm findings from miRNA array study, five miRNAs (miR-129, miR-135a, miR-191, miRNA-22 and miR-26b) were chosen to examine their expression in rat hippocampus by quantitative RT-PCR (qRT-PCR).

Other examples include miR-504 and miR-26b.

8

The overexpression of miR-26 in two human ATC-derived cell lines significantly decreased thyroid carcinogenesis, suggesting a crucial role for miR-26 down-regulation in thyroid carcinogenesis 29.

Thus, miR-26 exerts diverse effects on cellular function, either inhibiting or promoting cell proliferation in different cell types 24. miRNAs are evolutionarily conserved and act at the post-transcriptional level as “fine tuners” and/or “safeguards” to balance dramatic environmentally induced alterations in gene expression and maintain organism homeostasis 33.

MiR-26 may play crucial roles in growth and development of normal tissues and the pathogenesis of non-tumor diseases and tumor formation 24.

MiR-26 is overexpressed in high-grade glioma and is frequently amplified at the DNA level in a subset of human high-grade gliomas.

MiR-26, a functional miRNA, has received much attention from researchers in recent years.

These results indicate that the diverse effects of miR-26 on VSMC are species-specific and depend on the external stimuli.

9

miR-26b, which is regulated by glucose levels [47], is required for adipogenesis and target Pten and Adam17 [45, 46].

miR-26b is required for adipogenesis and target Pten and Adam17 [45, 46]; moreover, it is also regulated by glucose levels [47].

By contrast, miR-26b-5p, miR-199a-3p, miR-377–3p, miR-let-7f-5p, miR-200a-3p, miR-21–5p, miR-152–3p, and miR-192–5p expressions were repressed by SO diet consumption.

miR-26b-5p expression was lower after FO compared with PO diet.

Similarly, miR-215 expression decreased after FO compared to OO and PO diets and miR-26b-5p expression decreased after FO compared with PO diet and miR-9a-5p after FO compared with SO diet.

Likewise, we observed a decrease in the expression of several hepatic miRNAs, namely miR-192–5p, miR-10b-5p, miR-377–3p, and miR-215 after FO compared with OO and PO diets and miR-21–5p and mir-26b-5p after FO compared with PO diets.

10

Interestingly, the LII up-regulated miR-26b and miR-126 are implicated in Alzheimer’s disease (Absalon et al. 2013; Kim et al. 2014), while the LDeep up-regulated miR-219 and miR-7a/b are implicated in schizophrenia (Beveridge and Cairns 2012), diseases which both show pathologies in LII.

Several of the differentially expressed miRNAs regulate neuron differentiation, including miR-126 and miR-26b in LII, and miR-7a/b in LDeep.

11

Within the genetic background scanned for miRNA expression on Exiqon arrays, miR-24 and miR-26b were significantly correlated with GH and PRL, with miR-26b being reported to have a potential impact upon expression of the TF Pit-1 in GH3 cells by inhibiting the Pit-1 inhibitor called Lef-1 [59].

Correlation analysis shows the miR-26b expression is also related to GH and PRL transcript levels (Fig 6D), with relationships between this microRNA and TF’s discussed below.

12

The expression levels of miR-34a and miR-26b were significantly up regulated in serum of diabetic and IOMe -injected rats (Fig 5B), whereas the expression levels of these miRNAs are normally low.

The expression levels of miRNAs miR-34a and miR-26b were up-regulated in serum of diabetic and in IOMe-AG538 -injected rats (B).

13

For example, miR-26b up-regulates the growth hormone levels by targeting lymphoid enhancer binding factor 1 (Lef-1) in GH3 cells, whereas miR-129-5p, miR-202 and two other miRNAs repress the human growth hormone receptor (GHR) expression levels in both normal and cancer cells [17, 18].

For example, in 2010, Zichao Zhang et al. reported that miR-26b regulates two main factors, Lef-1 and pituitary-specific positive transcription factor 1(Pit-1), during pituitary development [17].

14

qPCR of left atrial chambers demonstrated that miR-1, miR-26b, miR-29a, miR-30e, miR-106b, miR-133 and miR-200 are up-regulated in HTD rats as compared to controls (Fig 1), demonstrating a similar microRNA expression profile as in atrial-specific Pitx2 deficient mice [14, 16].

Whereas it is wi dely documented that redox signaling can compromise ion channel functioning and calcium homeostasis in cardiomyocytes [67], in our system we observed no influence of H [2]O [2] administration on the regulatory impact of Pitx2 in distinct ion channels such as Scn5a, Kcnj2 and Cacna1c as well as multiple Pitx2-regulated microRNAs such as miR-1, miR-26, miR-29 and miR-200, in which redox impairment impact is less documented [68].

Several lines of evidence have already reported the key regulatory role of miR-1 [60– 62], miR-26 [63], miR-106b [64], miR-133 [65– 66] and miR-200 [64] in arrhythmogenesis.

Surprisingly none of the tested microRNAs, except for miR-26b and miR-106b, which in fact were decreased, display significant differences (Fig 2).

15

rno-miR-novel-8, rno-homolog-miR-26, and rno-homolog-miR-199 miRNAs were selected from Tier 1, and rno-miR-sno-57 miRNA was selected from Tier 2. In addition, we analysed the expression of miR-741-3p and miR-743a-3p and found that, in accordance with sequencing data, they were highly expressed in rat PSCs.

rno-homolog-miR-26 and rno-homolog-miR-199 miRNAs were expressed in EFs, ESCs and iPSCs, which is consistent with the data obtained from sequencing.

Four novel miRNAs with the following coordinates: chrX:−:151288045–151288101 (rno-miR-novel-8); chr7:+:70463555–70463594 (rno-homolog-miR-26); chr3:+:16697111–16697143 (rno-homolog-miR-199); and chr18:−:69422790–69422857 (rno-miR-sno-57) were selected for the validation.

16

The top 10 miRNAs upregulated and downregulated upon treatment with Tg and Tm, respectively are shown in Figure 2B-C. The expression of miR-98, let-7d*, miR-374, miR-181d, miR-352, miR-7a and miR-26b were increased both by Tg and Tm in H9c2 cells.

17

Among the miRNAs, miR-214, miR-199a-5p, miR-150, miR-199a-3p, miR-351, miR-145, miR-764, miR-497 and miR-92b were upregulated, whilst miR-7a, miR-325-5p, miR-485, miR-708, miR-344-3p, let-7f, miR-26b, miR-129, miR-29c and let-7a were downregulated.

These miRNAs include miR-214, miR-199a-5p, miR-150, miR-351, miR-145, miR-92b, miR-7a, miR-485, miR-708, let-7f, miR-26b, miR-129, miR-29c and let-7a.

18

Such a situation occurred for miR-26b, miR-30, and miR-374 downregulation, and for miR-34, miR-301, and miR-352 upregulation [121].

19

In addition, miR-26b was drastically downregulated in the high aggressive thyroid anaplastic carcinoma, whereas its levels did not change in the papillary and follicular histotypes, less aggressive thyroid carcinoma entities (57).

MiR-26 was downregulated in hepatocarcinoma (55) and colorectal carcinoma (56), and its loss was significantly linked to the metastatic phenotype.

20

Since the expression of LOXL2 is also influenced by hypoxia,, and microRNAs (miR-26 and mIR-29), there are also other potential strategies for targeting LOXL2 expression or activity (Wong et al., 2014).

21

Furthermore, miR-26b is involved in growth hormone (GH) regulation [12] and miRNAs have been identified as regulators of gonadotropins [13, 14].

For instance, miR-26b is involved in the development of the pituitary in mice [12].

22

Fish oil/pectin treatment up-regulated miR-19b, miR-26b and miR-203 expression as compared to corn oil plus cellulose (CCA) specifically in Lgr5 (high) cells.

23

Studies also showed that miR-21 [26], miR-26 [27], miR-328 [28], miR-133 and miR-590 [29] participated in the process of AF by controlling the expression of their specific gene targets.

24

Brain derived neurotrophic factor (BDNF) expression is regulated by microRNAs miR-26a and miR-26b allele-specific binding.

25

The upregulated miRNAs in the colon tissues of UC rats changed by HM were miR-149-5p, miR-351-5p, let-7d-5p, miR-98-5p, let-7a-5p, miR-3559-5p, let-7f-1-3p, miR-3596b, miR-224-5p, miR-411-3p, miR-184, miR-26b-3p, and miR-92b-3p.

26

Reports have shown that miR-208 and miR-140 affect their host genes 25, 26; however, miR-26 suppresses its host gene to regulate neurogenesis [27].

27

Furthermore, a recent study discovers that Huaier upregulates miR-26b-5p inducing cell apoptosis of pulmonary adenocarcinoma A549 cells [19].

28

miR that regulate targets in the mTORC1 pathway (hsa-miR-16-5p, hsa-miR-26b-5p, hsa-miR-99a-5p, hsa-miR-100-5p, hsa-miR-128a-3p, hsa-miR-133a-3p, hsa-miR-199a-3p, hsa-miR-221-3p) were analyzed using TaqMan® microRNA Assays (Applied Biosystems, Foster City, CA, USA).

29

Alike, miR-26b-5p influences translation of caveolin 2 and diacylglycerol O-acyltransferase 1, i. e. a key enzyme in hepatic steatosis in addition to lecithin-cholesterol acyltransferase which catalyzes its esterification for cholesterol transport.

30

Included among the 46 miRNAs with increased expression were 7 (miR-21, miR-16, miR-26a, miR-26b, miR-23a, miR-23b, miR-126) included in surveys of the most abundant miRNAs in human platelets [23, 24] and the miR-126 gene products miR-126-3p and miR-126-5p that are also enriched in vascular endothelial cells and endothelial microparticles [25].

31

2) Some miRNAs, including let-7 family (let-a, -b and -c), miR-16, miR-23b, miR-26, miR-31 and miR-375, were always highly expressed either before or after transdifferentiation (data not shown).

32

In general, the rank order of the expression levels of the measured miRNAs was comparable in mice and rats, with the highest miRNA levels of miR-16-5p and miR-223-3p and the lowest levels of miR-199a-3p, miR-652-3p, miR-423-3p and miR-26b-5p (S1– S3 Figs).

In AngII mice and controls, miR-26b-5p was significantly correlated to both LVESP (R = 0.66, P-value = 0.037) and dP/dt [max] values (R = 0.66, P-value = 0.038) and in mice with ischemic heart failure and controls we found miR-27a-3p to be borderline significantly correlated to LVEF (R = -0.56, P-value = 0.049).

33

The target miRNAs (and corresponding assay numbers) were: rno-miR-23a (000399), rno-miR-26b (000407), rno-miR-30-5p (000420), rno-miR-101b (002531), rno-miR-125b-5p (000449), rno-miR-379 (001138) and rno-miR-431 (001979).

34

Moreover, the endocannabinoid system has come under scrutiny given that several microRNAs such as miR26, miR146, and miR10 are responsible for its gene regulation [28, 29].

35

To facilitate the analysis of the array data, identical miRNAs of different species (e. g. bta-miR-126 and hsa-miR-126) were grouped together with miRNAs with the same precursor or closely related mature sequences (e. g. mdo-miR-26, hsa-miR-26a), as long as the seed sequences were still conserved.

36

On the other hand, a few miRs have been found to promote apoptosis of myocardiocytes, such as miR-26 [29], miR-34 [30], and miR-92 [31].

37

We designed and cloned into the pcPURhU6 vector the hairpin-type RNAs with si-6 sequence (pcPURhU6 si-6) with the 19-21 base pair (bp) stems and with various loops: (1) pcPURhU6 si-6 (21 bp)-miR26, (2) si-6 (19 bp) with 9-nt UUCAAGAGA loop [28], (3) si-6 (21 bp) with 9-nt UUCAAGAGA loop, (4) si-6 (21 bp) with 10-nt CUUCCUGUCA (loop from miRNA23), and (5) si-6 (21 bp) with 19-nt UAGUGAAGCCACAGAUGUA (loop from miRNA30) (see Figure 3).

38

MicroRNA profiling identified several miRNAs that have been previously associated with cardiac hypertrophy such as miR-214, miR-23b, miR-15b, rno-miR-26b, rno-miR-221, rno-miR-222, rno-miR-107 [59], miR-23a, miR-208, rno-miR-133b, miR-19a and mi-r133a [60].

39

MiR-26 and miR-328 control vulnerability of atrial fibrillation [15].

40

Type of site Context+ Context Structure Energy Is experimental validated rno-miR-344i MIMAT0025049 2 8mer −0.42 −0.334 297 −32.2 TURE rno-miR-6316 MIMAT0025053 2 8mer, 7mer-m8 −0.41 −0.611 308 −29.59 TRUE rno-miR-21-3p MIMAT0004711 2 8mer, 7mer-m8 −0.408 −0.581 289 −25.18 TRUE rno-miR-3120 MIMAT0017900 2 7mer-m8 −0.402 −0.536 289 −24.4 TRUE rno-miR-194-5p MIMAT0000869 3 7mer-m8 offset 6mer −0.381 −0.593 442 −41.91 TRUE rno-miR-126a-3p MIMAT0000832 1 8mer −0.358 −0.248 148 −18.86 TRUE rno-miR-27a-3p MIMAT0000799 3 7mer-m8 −0.357 −0.708 447 −41.04 TRUE rno-miR-26b-5p MIMAT0000797 3 7mer-m8 offset 6mer −0.348 −0.581 444 −30.64 TRUE rno-miR-3557-3p MIMAT0017820 4 8mer 7mer-m8 imperfect −0.346 −0.503 582 −81.21 TRUE rno-miR-27b-3p MIMAT0000798 4 7mer-m8 offset 6mer −0.334 −0.705 588 −55.75 TRUE rno-miR-3569 MIMAT0017849 2 8mer offset 6mer −0.333 −0.